Anisotropic rotational motion of membrane proteins will be studied. A pair of hydrophobic, bifunctional spin label reagents will be used to determine the orientation of the probe, with respect to the axis of rotation of the protein in the membrane. The two labels will differ by having the nitroxide principal Z-axis rotated by 90 degrees relative to the molecular long axis. The proteins to be studied are bacterio-rhodopsin, erythrocyte band 3 and sarcoplasmic reticulum ATPase. The rate of rotational motion of the proteins will be determined by saturation transfer electron paramagnetic resonance. The evaluation of anisotropic motion of spin labeled membrane proteins will be assisted by a detailed study of slow anisotropic motions of spin labels in model systems. The motion of both 14N and 15N spin labels trapped in thiourea crystals will be analyzed. The aim is to determine which spectral parameters are good indicators of anisotropic, as opposed to isotropic, motion. Conditions for attaching substrates and inhibitors of cell surface enzymes or receptor through the membrane, to spectrin, or other submembranous filaments, will be investigated. Molecules which may serve as substrates for membrane-bound protein kinases will be prepared. The object is to find a molecule that will give a spectral change when it is phosphorylated in an intact cell. A new class of transmembrane reagents will be investigated. This class will be based on gramicidin A, a transmembrane antibiotic.